This project concerns the regulation, in vitro and in vivo, of the rate-limiting catalyst for cholesterogenesis, HMG-CoA reductase (reductase). Topics to be investigated include the structure of the reductase regulatory (phosphorylation) site, the stoichiometry of phosphate:reductase monomer, purification and characterization of the reductase converter proteins reductase kinase and reductase phosphatase, in vivo labelling of reductase and reductase kinase in a hepatocyte system and changes in labelling rates in response to physiological effectors, determination of whether reductase undergoes changes in modulation state in vivo (hepatocytes), characterization of the kinetic parameters of homogeneous mammalian reductase, verification or revision of the proposed model for catalysis, and determination of the effect of phosphorylation on catalysis. We wish to answer these questions: 1) When reductase is phosphorylated, to what is phosphate attached? 2) How many moles of phosphate per mole of reductase monomer suffice to inactivate catalytic activity? 3) What is the primary structure of the phosphorylation site? 4) Can we employ purified reductase kinase to prepare 32p reductase for use in a 32p-release assay for reductase phosphatase and can we develop a 32p assay for reductase kinase? 5) Are reductase kinase and reductase phosphatase specific for their substrates? 6) Do reductase, reductase kinase and/or reductase phosphatase undergo changes in activity in response to addition of hormones and related compounds to hepatocytes? Is the yeast and Pseudomonas model for catalysis by reductase valid for mammalian reductase? 8) What portion of the multi-step reaction mechanism for reductase is most affected when reductase is phosphyorylated? Techniques: 32p Labelling, enzyme purification, rapid kinetic assays, hepatocytes.